The spore formation and toxin production in biofilms of Bacillus cereus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

Listed in Dean's List of Exceptional Theses 2022Bacillus cereus (B. cereus) is a foodborne pathogen causing diarrhoea and emesis which are the consequences of enterotoxin and emetic toxin production, respectively. Sporulation and biofilm formation are used as survival strategies by B. cereus protecting cells from harsh environments. However, these survival strategies also make B. cereus more difficult to control in the food industry. The aim of this study is to investigate the spore formation and toxin production in the biofilm of B. cereus. In this study, higher sporulation and higher spore heat resistance were demonstrated in biofilms grown on stainless-steel (SS) compared to planktonic populations. The structure of coat in spores isolated from biofilms, the upregulated germination genes in planktonic cells and upregulated sigma factor B in biofilm cells are possible explanations for these observations. The levels of dipicolinic acid (DPA) did not affect the heat resistance of spores harvested from biofilms in this study. Haemolytic toxin (Hbl) was mainly secreted by cells into surrounding media while emetic toxin (cereulide) was associated with cells. Higher Hbl toxin was observed in the presence of biofilms grown on SS compared to either planktonic culture or biofilm grown on glass wool (GW) using the Bacillus cereus Enterotoxin Reverses Passive Latex Agglutination test (BCET-RPLA). This was supported by the significant (P < 0.05) increase in HblACD expression in biofilm cells on SS, using both real-time quantitative PCR (RT-qPCR) and RNA sequencing. The transcriptomic analysis also revealed that biofilms grown on SS had an upregulated secretion pathway, suggesting biofilms of B. cereus grown on SS are more pathogenic than planktonic cells. Unlike the Hbl toxin, cereulide was associated with biofilm cells/structures and attached to the biofilm-forming substrates including SS and GW used in this study. The expression of cerA and cerB was similar between biofilms and planktonic cells using RT-qPCR. This project highlights the importance of biofilms by B. cereus in food safety through the enhanced heat resistance of spores, the higher Hbl toxin production and attached cereulide toxin

A single-cell view on the intra- and inter-population metabolic heterogeneity and ecophysiology of microorganisms at different ecological scales

Metabolic heterogeneity (MH) occurs when isogenic microbial populations display cell-to-cell differences in metabolic traits, albeit exposed to homogeneous conditions. Despite the increasing focus on MH, its triggering factors remain largely unknown. In the present thesis, I used stable isotope probing and chemical imaging with nanoscale Secondary Ion Mass Spectrometry (nanoSIMS) to study MH at single-cell level, in model organisms, synthetic and natural communities, to understand i) how abiotic factors, biotic interactions and antibiotics exposure influence MH and ii) its potential ecological role. Moreover, I optimized sample preparation for chemical and high-resolution imaging and suggested two different indices as ‘unit measure’ of MH. As results, I have shown for the first time that MH is displayed by microorganisms under favorable growth conditions, although none of the tested abiotic factors prevailed as the main trigger of MH. I brought insights on how biotic interactions play a role in the functional heterogeneity using bacteria pseudo-fungi co-cultures. I found that antibiotics reduce Carbon and Nitrogen assimilation rates of targeted phylogenetic groups in river-water communities, while increasing their MH, pointing to its ecological importance in natural environments. To conclude, I provided novel insights on the phenomenon of MH and its dynamics at different ecological scales.:Abbreviation list Summary Introduction Knowledge gaps Results and discussion - Optimization of sample preparation - Validation of quantitation methods - Abiotic factors shaping metabolic heterogeneity in bacterial populations - Influence of biotic factors in shaping heterogeneity - Metabolic Heterogeneity and ecophysiology of natural microbial populations influenced by emerging contaminants Conclusions Outlook Bibliography Appendix Acknowledgments Curriculum Vitae List of publication

Studies in bacterial genome engineering and its applications

textMany different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems.Cellular and Molecular Biolog